In vitro evaluation indicated that the mixed micelle was an ideal PTX delivery system for the treatment of MDR tumors; the mixed micelle also showed a significantly better drug-loading coefficient than Genexol-PM.Genexol-PM, produced by Samyang Company (Korea) is an excellent preparation of paclitaxel (PTX) for clinical cancer treatment. However, it cannot resolve the issue of multidrug resistance (MDR)—a significant problem in the administration of PTX to cancer patients. To increase the efficacy of Genexol-PM against MDR tumors, a mixed micelle capable of serving as a vehicle for PTX was developed, and two substances were chosen as carrier materials: 1) Polyethylene glycol–polylactic acid (PEG-PLA), the original vehicle of Genexol-PM. 2) Vitamin E-TPGS, an inhibitor of P-glycoprotein (P-gp). P-gp has been proven to be the main cause of MDR.  Tumors are a great threat to human health, and paclitaxel (PTX) is one of the most important anticancer drugs in clinical use today. PTX inhibits microtubule depolymerization of free tubulins; and it has shown its potency against a variety of tumors ,2. HowevVarious drug delivery systems have been developed to replace the vehicle of Taxol to overcome the aforementioned limits. These new delivery systems are mostly aqueous media, such as polymeric micelles ,5. GenexTo retain the advantages of Genexol-PM, its PEG-PLA vehicle had to be preserved and another amphiphilic block copolymer capable of overcoming the MDR and had properties similar to those of the PEG-PLA had to be found. Previous studies have shown that some pharmaceutical excipients can reverse the MDR property of tumors ,16, inclin vitro.The purpose of this study is to develop a mixed micelle composed of PEG-PLA and Vitamin E-TPGS, incorporating PTX in the core. It may improve the anticancer efficacy by overcoming the MDR of tumor cells. The PTX-loaded mixed micelle was prepared using a thin-film hydration method and its formulation was optimized. Tumor cells were used to verify the improvement PTX was purchased from Xi’an Sanjiang Bio-Engineering Co., Ltd. . Methoxy poly (ethylene glycol)  was obtained from JenKem technology Co., Ltd. . Vitamin E-TPGS was obtained from Peboc . Coumarin 6, Vinblastine, Tween 80, and 3--2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma–Aldrich, Inc. . Hoechst 33342 and Lyso Tracker Red were purchased from Beyotime Biotechnology Co., Ltd. . Cellulose ester membranes  with a molecular weight cut-off value (MWCO) of 3500 Da  were used in the dialysis experiments. Acetonitrile and methanol were supplied by Burdick and Jackson Corp. . Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640, fetal bovine serum (FBS), Penicillin-streptomycin, and a 0.25% (w/v) trypsin–0.03% (w/v) EDTA solution were purchased from Gibco BRL . Deionized water was prepared with a Millipore water purification system . Acetonitrile was High Performance Liquid Chromatography (HPLC) grade, and all other chemicals were reagent grade and used without further purification.2 under fully humidified conditions, supplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. A549 was cultured in DMEM; KB and KBv were cultured in RPMI 1640. In addition, the KBv culture was supplemented with vinblastine in the medium, the final concentration of which was 200 ng/ml.The human lung adenocarcinoma cell line A549, human oral epithelial cancer cell line KB, and its vinblastine-resistant subclone KBv were obtained from the Cell Resource Center of the China Science Academy, Shanghai, China. All cells were cultured at 37°C with 5% CO2000-PLA, each with a different molecular weight, were synthesized  . These were calculated as follows.The amphiphilic block ratio of the PEG-PLA is critical to the micelle formation even for mixed micelles . Three k, 38/62) . The encThe PTX-loaded mixed micelles were prepared by a thin-film hydration technique . BrieflyAfter a series of preliminary experiments to assess the control factors and their corresponding levels, the VE-TPGS mass fraction and the amount of PTX fed in were selected as two variables at five levels and used to optimize the formulation process; particle size, EE%, DL%, and the relative amount of PTX precipitated from micelles after 24 h of constant temperature oscillation at 37°C (PTX Pre%) were taken as four responses. PTX Pre% value was calculated using the following equation:The effects of the variables were studied with a central composite design (CCD) ,28, and The mean particle size and zeta potential of the PTX-loaded PEG-PLA micelle and the PTX-loaded mixed micelle were measured using dynamic light scattering (DLS) analysis with a Zeta Potential and Particle Sizer . The morphological examination of micelles was performed under a transmission electron microscope  after negative staining with a phosphotungstic acid solution .2 standard solution added. The UV absorbance values of the polymer solutions were measured at 366 nm with a UV-VIS spectrometer . The intensity of absorption was plotted against the logarithm of the polymer mass concentration.The critical micelle concentrations (CMC) of PEG-PLA, VE-TPGS, and their mixture  were determined using iodine UV spectroscopy as described previously . A set oTo investigate the incorporated form of the PTX and the polymers, approximately 3 mg of PTX, blank mixed micelle, and PTX-loaded micelle were sealed separately in standard aluminum pans with lids, and subjected to differential scanning calorimetry . The temperature ramp speed was set at 10°C/min (from 100 to 300°C).in vitro, phosphate buttered saline  with 0.5% Tween 80 was chosen as the medium to create a pseudo-sink environment [In order to compare the release behaviors of PEG-PLA micelles and mixed micelles ironment . One mic18, 200 mm × 4.6 mm, Phenomenex, CA, U.S.), and an SPD-10AVP UV detector ; and operated at 230 nm. The mobile phase was a mixture of acetonitrile/ water . The flow rate was maintained at 1 ml/min. The PTX concentrations were determined by comparing the peak areas with the standard curve.The concentration of the PTX in the samples was determined with the HPLC. The machine was a Shimadzu HPLC system equipped with an SIL-10AF auto sampler, an LC-15C pump, a reversed-phase column .Coumarin-6 was chosen as a fluorescence probe loaded with micelles for assessment of cell uptake. The method of preparation described in 5 cells/well. After incubation with coumarin-6-loaded micelles, as described above, the cells were trypsinized and centrifuged at 2000 rpm for 4 min to obtain a cell pellet, which was subsequently resuspended in PBS and analyzed with a flow cytometer .For quantitative analysis, A549 cells were seeded into 6-well plates at the density of 1×10Triple-labeling was made to determine which sub-cellular organelles were involved in the cytoplasmic distribution of the micelle. The localization of coumarin-6-labeled micelles was visualized with fluorescence markers specific to organelles, including LysoTracker Red and Hoechst 33342. A549 cells were seeded into a glass-bottom culture dish and cultured for 24 h and then incubated with 200 μg/ml of coumarin-6-loaded PEG-PLA micelles and coumarin-6-loaded mixed micelles. Then the cells were further treated with 50 nM LysoTracker Red (30 min) and 10 μg/ml of Hoechst 33342 (10 min) to visualize the lysosomes and nuclei, respectively. Finally, the cells were washed three times with cold PBS and observed under a laser scanning confocal microscope (Leica TCS SP5).3 cells/well into 96-well plates. After 24 h incubation, the media were substituted with different PTX formulations at a series of concentrations (0.001 to 10 μg/ml) for 72 h, including the PTX solution (1.0% DMSO in PBS), PTX-loaded PEG-PLA micelles, and PTX-loaded mixed micelles. Cell viability was measured with an MTT assay and the absorbance of each well was determined at 570 nm with a microplate reader . To evaluate the effect of the excipients, the cytotoxicity of the blank mixed micelle was also tested with the method described above at concentrations ranging from 0.1 to 1000 μg/ml. The results are here expressed as percent cell viabilities relative to that of cells incubated in the culture medium without drugs or excipients.To measure the cytotoxicity of the two PTX formulations and evaluate the role of the VE-TPGS in reversing MDR, A549 cells, KB cells, and KBv cells were seeded at a density of 5×104 cells/well, gently shaken for 5 min, and incubated at 37°C for 7 days. Uniform and compact multicellular spheroids were chosen for subsequent studies.An A549 tumor spheroid model was built with the lipid overlay method as reported previously ,32. Briein vitro and to confirm the effect of the mixed micelles on overcoming MDR, the tumor spheroids were incubated with the coumarin-6-loaded PEG-PLA micelles and the coumarin-6-loaded mixed micelles, diluted by a DMEM medium to the micelle concentration of 200 μg/ml at 37°C for 4 h. Then, the tumor spheroids were rinsed with cold PBS and fixed with 4% paraformaldehyde before subjection to a laser scanning confocal microscopy analysis .To compare the tumor-penetrating ability of the PEG-PLA micelle and the mixed micelle on solid tumors P < 0.05.The results are here given as mean ± standard deviation. The statistical difference between the two mean values was calculated using a two-tailed Student’s t-test. The values were considered statistically significant if 2000-PLA may have a block ratio optimal for incorporating PTX, especially when maximum EE% and DL% are required. At a low feeding ratio (2:50), the PTX-loading capacity of the three groups did not differ significantly. As the feeding ratio increased, the EE% of groups 1 and 3 both dropped significantly, and the DL% of groups 1 and 3 were significantly lower than theoretical values. These results suggested that group 2 , in which the amphiphilic block ratio was close to 50/50, produced the best drug-loading capacity. The ratio was also similar to that observed in research on Genexol-PM [As reported previously, increasing the chain length of the hydrophobic block may result in a significant increase in the hydrophobic drug-loading capacity of the copolymer . CompareThe numerical optimization module in Design-Expert was used to search for a combination of factor levels that satisfied the requirements placed on each of the responses and factors. For better drug-loading capacity and micelle stability, the requirements were set to maximize the EE% and DL%, and minimize the particle size and PTX Pre%. Through a desirability function, the module predicted the ranges of variables where the optimal formulation may occur . The optThe mean diameters and zeta potentials of the PEG-PLA micelles and mixed micelles were 22.46±0.54 nm, -4.36 mv and 16.36±0.78 nm, -2.79 mv, respectively. 3− to I2, which maintained the saturated concentration of I2 solution [Iodine was used as a hydrophobic probe to monitor the formation of the mixed micelles. Because it is a small hydrophobic molecule, iodine was incorporated into the hydrophobic cores of the micelles. This caused a conversion of Isolution . The CMCsolution .The encapsulation of PTX into the core of the mixed micelle was confirmed by DSC. in vitro release profile of the mixed micelle was similar to that of the PEG-PLA micelle, suggesting that the addition of VE-TPGS as part of the vehicle did not influence the in vitro release behavior of the PTX-loaded PEG-PLA micelle. The release behavior of the PTX from the mixed micelle exhibited a biphasic pattern with a characteristic fast initial release during the first 3 h, followed by a slower, continuous release. Accordingly, the in vitro release profile of the PTX from the polymeric micelle is largely affected by the hydrophobic properties of its inner core [As shown in ner core . The strP<0.05)  . The resin vitro was evaluated on A549 cells. As shown in 50 was still greater than 1 mg/ml, which meant that the PEG-PLA and the VE-TPGS were both biocompatible and that the mixed micelle could be used safely to deliver PTX.The biocompatibility of the materials 50 values as follows: mixed micelle < PEG-PLA micelle < Free PTX (P<0.01). This showed that the mixed micelle group had a significantly greater cytotoxic effect on A549 cells.The cytotoxic effect of the PTX-loaded micelle was determined employing the A549 cell line, at the beginning . A549 ceFree PTX . There w50 values were 0.05 μg/ml for the PEG-PLA micelles and 0.03 μg/ml for the mixed micelles ; this observation was consistent with the results regarding the A549 cell line. These results demonstrated that the addition of VE-TPGS could help the PEG-PLA micelle enhance the cytotoxicity against MDR cancer cells. The overexpressed P-gp in A549 and KBv cells led to increased drug efflux, and PTX has been confirmed to be a P-gp substrate [To confirm the effect of the VE-TPGS on the result by its function in reversing the MDR of A549 tumor cells, MTT assays of the human oral epithelial cancer cell line KB and its vinblastine-resistant subclone KBv were designed. KB cells, which were sensitive to PTX, were used as control, to compare with the MTT assay results of the KBv cells. After 72 h of incubation, the PEG-PLA micelles and the mixed micelles both exhibited a strong cytotoxicity against the proliferation of KB cells . The IC5micelles . Howevermicelles . Unlike ubstrate ,14. As rubstrate . This miin vivo and evaluate the penetrating ability of the micelles on solid tumors. As shown in Three-dimensional multicellular tumor spheroids of the A549 cells were established to mimic the solid tumors 2000/PLA = 54/46 is the best option. A CCD was used to optimize the formulation: The result was a mixed micelle with a particle size of around 16.36 nm. Surprisingly, compared with Genexol-PM, the mixed micelle improved the PTX-loading capacity significantly (DL% = 24.32%) and had a lower CMC, i.e., a stronger anti-dilution capacity. For MDR tumor cells , PTX-loaded mixed micelle displayed noticeable antitumor efficacy. It enhanced the cell uptake significantly by overcoming MDR. These results showed that the PEG-PLA/Vitamin E-TPGS mixed micelle may be better than Genexol-PM as a vehicle of PTX in the treatment of MDR tumors.In this study, we developed a new mixed micelle to improve the antitumor efficacy of Genexol-PM. The new micelle consists of PEG-PLA  and VE-TPGS. This micelle is a suitable vehicle for the poorly soluble anticancer drug PTX. VE-TPGS was chosen for its ability to overcome the MDR of tumor cells. The amphiphilic block ratio of PEG-PLA was found to be critical to the mixed micelle formation; we think that the ratio PEGS1 File(PDF)Click here for additional data file.


This study aimed to investigate the dosimetric characteristics of an isocentrically shielded RapidArc (IS-RA) technique for treatment of locally recurrent nasopharyngeal cancer (lrNPC). In IS-RA, the isocenter was placed at the center of the pre-irradiated brainstem (BS)/spinal cord (SC) and the jaws were set to shield the BS/SC while ensuring the target coverage during the whole gantry rotation. For fifteen patients, the IS-RA plans were compared with the conventional RapidArc (C-RA) regarding target coverage, organ-at-risk (OAR) sparing and monitor units (MUs). The relationship between the dose reduction of BS/SC and some geometric parameters including the angle extended by the target with respect to the axis of BS/SC (Ang_BSSC), the minimum distance between the target and BS/SC (Dist_Min) and the target volume were evaluated. The IS-RA reduced the BS/SC doses by approximately 1–4 Gy on average over the C-RA, with more MUs. The IS-RA demonstrated similar target coverage and sparing of other OARs except for slightly improved sparing of optic structures. More dose reduction in the isocentric region was observed in the cases with larger Ang_BSSC or smaller Dist_Min. Our results indicated that the IS-RA significantly improves the sparing of BS/SC without compromising dosimetric requirements of other involved structures for lrNPC. Because this was not a treatment-based study, our institutional review board waived the need for written informed consent from the participants. The patient information was anonymized and de-identified to protect patient confidentiality. The methods were carried out in accordance with the approved guidelines.Fifteen lrNPC patients with Stage rT1–rT4, N0-1, M0 were included in this study, staged according to the American Joint Committee on Cancer (AJCC) 7th edition staging system. Eleven were male and four were female, with the median age of 49  years. All the patients received radical chemoradiation in the first treatment and the median time to treatment failure was 16 months .All of the patients were immobilized in the supine position in a tailor-made thermoplastic cast from head to shoulders. CT scans with intravenous contrast using a 3 mm slice thickness from the head to 2 cm below the sternoclavicular joint were performed by a CT scanner . The CT images were then transferred to the Eclipse (version 10.0) treatment planning system  for target and OAR delineation and treatment planning.3 with the range of 38–209 cm3.The gross tumor volume (GTV) included the recurrent primary lesions and positive lymph nodes, which were determined by the CT, MRI, Positron Emission Tomography (PET) and endoscopic findings. Clinical target volume (CTV) encompassing microscopic disease was defined as the GTV plus margins of 8–10 mm, allowing smaller margins close to critical intracranial structures or the SC. Planning target volume (PTV) was generated to account for setup variability and internal motion by adding 3 mm margins to the CTV. The median volume of the PTV (Vol_PTV) was 89 cmThe OARs, including the SC, BS, lenses, optic nerves, optic chiasm, temporomandibular (T-M) joints, temporal lobes, oral cavity and parotids were contoured. Planning organ-at-risk volumes (PRVs) were created by adding 5 mm margins to the SC and 3 mm margins to the BS, denoted as PRV-SC and PRV-BS, respectively. Normal tissue was defined as the body subtracting the PTV.Three different plans were created for each patient in Eclipse using the three different techniques, IS-RA, conventional RA (C-RA) and RA with the same gantry and collimator angles as those of IS-RA (RA-SGC). 6-MV photon beams from the TrueBeam linear accelerator  were employed for all the plans. The Progressive Resolution Optimizer  algorithm was used for RA optimization. The prescribed dose to the PTV was 60–69.9 Gy (2.00–2.33 Gy/fraction) administered in 30 fractions. All the treatment plans were normalized to achieve the goal of 95% of the PTVs covered by 100% of the prescription dose, except for 4 advanced cases where compromises were necessary to protect the critical OARs.x represents the dose which is reached or exceeded in x of the volume. V100% represents the % volume covered by 100% of the prescription dose. The planning constraints were fine-tuned, balancing the tradeoffs between the lowest dose to the BS/SC and the acceptable PTV coverage.The planning goals of the PTVs and OARs used in this dosimetric study are shown in In generating an IS-RA plan, the isocenter was set at the center of the BS (13 cases) or SC (2 cases), to which the PTV was adjacent. Four or six coplanar partial arcs were placed in two gantry rotations . The collimators rotated to angles approximately parallel to the BS and SC during the gantry rotation, generally 1°–20°. The jaw positions were set individually for each partial arc, with the priority of shielding the PRVs of BS and SC, followed by the partial coverage of PTV, but ensuring full PTV coverage if possible during the whole gantry rotation. The geometry settings of a representative IS-RA plan are shown in For each C-RA plan, the isocenter was set at the center of the PTV. Two coplanar full arcs (gantry angle: 181°–179° and 179°–181°) with the same non-coplannar arcs settings as IS-RA were used. The collimator angles were set to 30° aiming at minimizing the tongue and groove effect. The optimization objectives of the C-RA plans were set the same as those of the IS-RA plans.In order to discriminate the effects of the isocenter placement in the BS/SC and the prolonged arc length with different collimator rotation (1°–20°), the RA-SGC plans were generated, in which the only differences from IS-RA were that the isocenter was placed at the center of the PTV (but not in the BS/SC) and the jaws were automatically set to fit to the whole PTV . The same optimization objectives were used. All the plans were conducted by a medical physicist.2% and D98% were selected as near-maximal and near-minimal doses for the PTV, respectively. Homogeneity index (HI) was employed to assess the target dose homogeneityTo compare the three plans, dose-volume statistics, isodose distributions and cumulative dose-volume histograms (DVHs) were calculated. According to the International Commission on Radiation Units and Measurements (ICRU) report 83, DThe target dose conformality was measured by the conformity index (CI) introduced by Paddick5% was used to evaluate the dose received by the most heavily irradiated 5% volume of the organ9152%, D5% and mean dose (Dmean) were used for evaluating the doses delivered to the OARs. The geometric parameters of the PTV and its geometric relationship with other structures, including the angle extended by the PTV with respect to the axis of BS/SC (Ang_BSSC)  , the minP-value of <0.05 was considered to be statistically significant.Statistical analyses were performed using the SPSS (version 19.0) software . The comparison among the IS-RA, C-RA and RA-SGC plans were tested with two-sided Wilcoxon signed rank test. The effect of the geometric parameters of the PTV  on the BS/SC sparing was investigated using linear regression analysis. 100% of PTV was equal to 95%. In the remaining 4 advanced cases (12 plans), the V100% of PTV was less than 95%, with the lowest value of 88.4%. The doses of all the OARs were limited to the tolerable levels.A systematic approach referred to as IS-RA was established for the treatment of lrNPC. In most plans (33 out of 45 plans), the V2%. Data of target coverage for all 45 plans is summarized in 2%/D5%/Dmean of the (PRV of) BS by 3.3–3.8 Gy/3.5–3.7 Gy/2.4–2.6 Gy on average. The D2%/D5%/Dmean of the (PRV of) SC was reduced by 2.4–2.8 Gy/2.2–2.5 Gy/0.9–1.1 Gy on average with IS-RA technique. These reductions of BS/SC doses were statistically significant  BS became more obvious. However, the level of SC sparing decreased with minor reduction of D2%/D5% of the PRV-SC as the Ang_BSSC increased or as the Dist_Min decreased. In the present case (P > 0.05).The relationship between the Ang_BSSC/Dist_Min/Vol_PTV and the dose reductions of the (PRVs of) BS/SC by IS-RA are demonstrated in ent case , 3 and 42% when compared to C-RA. IS-RA also reduced Dmean to the normal tissue by up to 0.16 Gy compared to both C-RA and RA-SGC (P < 0.05). The doses to most other OARs were comparable among the three plans.It is also noticed that IS-RA exhibited slightly superior sparing of the optic structures, by up to 1.26 Gy in terms of DAdditionally, the IS-RA produced 40.1 ± 18.9% and 34.9 ± 20.1% more MUs than the C-RA and RA-SGC plans, respectively.The proposed IS-RA addresses an unmet need in treatment of lrNPC, as it substantially reduces the doses to the BS and SC while adequately covering the PTV. In general, the BS and SC are proximal to the locally recurrent lesions and their threshold doses have often been reached in the primary treatment course, making it challenging to irradiate the recurrent tumor(s) without exceeding the dose limit of the BS and SC. It is technically difficult to reduce even 1-Gy dose to BS/SC, especially in the challenging cases. As compared to C-RA, the IS-RA produces a sharper dose gradient between the PTV and the BS/SC, leading to an improved BS/SC sparing of approximately 1–4 Gy on average  with simultaneously improved or comparable dose to the PTV and other structures. The efficacy makes IS-RA a useful tool in clinical practice, because reducing the dose to BS and SC to the great extent  in re-irradiation course may reduce the life-threatening risk of BS necrosis and myelitis. It is noteworthy that the IS-RA technique also reduces the doses to the optic nerves, optic chiasm and lenses, which may be beneficial in reducing the risk of radiation-induced optic complications, such as blindnessBy comparing with RA-SGC, we confirmed that the superior performance of the IS-RA in sparing the BS/SC is mainly attributed to the isocentrically-shielded effect of the OARs with the collimator, not the prolonged arc length or different collimator angles. In geometry, the IS-RA technique focused the beam-shielded projection on the isocentric region during the whole treatment, so the dose of isocentric region could be minimized. During the entire treatment process, the beams across the BS/SC in the isocentric region are avoided, which receives only the scattered radiation from the nearby areas within the open parts of beamset al.5% of 12.83 ± 1.72 Gy and 2.18 ± 1.17 Gy, respectively, while the D5% of BS and SC in photon IMRT were 19.47 ± 1.01 Gy and 13.62 ± 2.17 Gy. The proton beam therapy owes its successful dose sparing to the Bragg peak. However, the proton accelerator is not widely available nowadays. In contrast, the IS-RA technique is capable of producing a comparable D5% of BS and SC using commonly used photon beams, as indicated in et al.et al.et al.There are a few related techniques for lrNPC that are worth of mentioning. Liu One limitation of the proposed technique was the increased MUs, which may increase the risk of secondary cancers in theory due to leakage radiation to patients232425The proposed isocentrically shielded RapidArc technique takes advantages of desirable features of rotational arc delivery and central sensitive structure blocking strategy and provides much better sparing of the pre-irradiated brainstem and spinal cord without compromising dosimetric requirements of other organs at risk and the PTV. The technique provides a viable choice for the re-irradiation of locally recurrent nasopharyngeal cancer.How to cite this article: Lu, J.-Y. et al. Dosimetric analysis of isocentrically shielded volumetric modulated arc therapy for locally recurrent nasopharyngeal cancer. Sci. Rep.6, 25959; doi: 10.1038/srep25959 (2016).


Prostate cancer and chronic lymphocytic leukemia (CLL) are relatively common malignancies associated with advanced age. Although immunotherapy-based strategies are used to treat both, currently, there is no overlap in specific therapies. Sipuleucel-T is an active cellular immunotherapy that improves overall survival for patients with metastatic castration resistant prostate cancer (mCRPC) but is not typically associated with a decline in prostate-specific antigen (PSA) following administration. We report the case of a 78-year-old man with mCRPC and Rai stage 0 CLL who sustained a 12-month decline in both PSA and white blood cell (WBC) count following treatment with APC8015-2 , as part of the phase II ProACT clinical trial. Two years later, the patient received commercial sipuleucel-T and again was noted to have a decline in PSA. Exploratory analysis did not clearly identify any peripheral immune markers associated with response. This case report suggests that treatment with sipuleucel-T can rarely lead to PSA decline, may have dual activity against both prostate cancer and CLL, and that these findings warrant further investigation. Prostate cancer is the most common non-skin malignancy affecting men and is responsible for the majority of cancer deaths in men in the United States. The first-line treatment of metastatic prostate cancer includes androgen deprivation therapy (ADT), which leads to a varying period of disease control. Eventually, the disease progresses despite ADT, representing the castration resistant disease state. Treatment options for metastatic castration resistant prostate cancer (mCRPC) in the past decade have expanded greatly, with five new agents recently approved by the Food and Drug Administration (FDA), including the first-in-class autologous cellular immunotherapy, sipuleucel-T (Provenge®) , 2.Sipuleucel-T is produced by ex vivo activation of patient-derived peripheral blood mononuclear cells by incubation with a fusion protein, PA2024, comprised of granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostatic acid phosphatase, which is expressed on over 95% of prostate cancers . The resChronic lymphocytic leukemia (CLL) is the most common leukemia diagnosed in the United States and Western world. CLL is a B cell chronic lymphoproliferative neoplasm that occupies one end of a single disease spectrum defined by the WHO with small lymphocytic lymphoma (SLL), a mature, peripheral B cell neoplasm, at the other end. Like prostate cancer, CLL is a disease associated with older age as well as with a highly variable clinical course ranging from indolent disease requiring no treatment to aggressive, lethal disease , 5. The We report the case of a patient with mCRPC who also had Rai stage 0 CLL. He was initially treated with APC8015-2  as part of a clinical trial and later with commercial sipuleucel-T.The patient was initially diagnosed with localized Gleason 7 prostate cancer in 1989, underwent radical prostatectomy in 1990, and received salvage radiation in 1994, followed by an undetectable prostate-specific antigen (PSA).In December of 2005, the patient had an episode of gross hematuria and underwent transurethral resection of bladder tumor for presumed bladder cancer. He subsequently underwent cystectomy on March 8, 2006 with pathology unexpectedly showing metastatic prostate adenocarcinoma, Gleason 4+5. Post-cystectomy, PSA was 28.48 ng/mL on March 28, 2006, and he was started on ADT on May 2, 2006. He reached PSA nadir of 0.19 ng/mL in November 2006, which was followed by a slowly rising PSA, representing castration-resistant disease.In November of 2008, 2 years after initiation of ADT, PSA rose to 7.35 ng/mL. He was then started on ketoconazole 400 mg PO daily, which was discontinued in April 2009 (PSA 3.93 ng/mL) due to diarrhea attributed to the ketoconazole. PSA declined to a nadir of 0.04 ng/mL in August 2009 and later began to rise again.PROstate Cancer with Active Cellular ImmunoTherapy (ProACT) dosing study of sipuleucel-T  in men with mCRPC. In this study, eligible patients were randomized to receive either sipuleucel-T, manufactured using the FDA-approved standard 10 μg/mL concentration of PA2024, or a product manufactured in a similar manner to sipuleucel-T but using a lower concentration of PA2024: 5 μg/mL (APC8015-5) or 2 μg/mL (APC8015-2).In June of 2010, the patient’s PSA increased to 9.08 ng/mL. Restaging with CT and bone scans revealed a new bone lesion in the right sixth rib. He was enrolled in the phase II treatment of 9 cells/L and PSA of 23.68 ng/mL at time of starting study  count of 17.21 × 10The patient received three study infusions of study drug with the final dose administered on December 2, 2010. Subsequent unblinding revealed that he had been randomized to receive APC8015-2, the investigational form of sipuleucel-T produced using 2 μg/mL of PA2024. Correlative markers drawn for the ProACT study demonstrated that CD54 upregulation (a measure of product potency and antigen-presenting cell activation [9 cells/L with an absolute lymphocyte count (ALC) of 5.01 × 109 cells/L, despite the fact that the patient was not receiving concurrent treatment for CLL; ALC was not consistently measured and is reported when available. This suggested that the APC8015-2 he received as part of the ProACT study rendered control of not only his prostate cancer but potentially also his CLL.Approximately 6 months after study treatment, PSA had declined from 23.68 to a nadir of 0.25 ng/mL on May 4, 2011. In addition, WBC declined from 17.21 to 9.45 × 10By April 16, 2012, the patient’s PSA, WBC, and ALC rose to 6.53, 9.75, and 5.55 ng/mL, respectively. He was subsequently retreated with commercial sipuleucel-T (produced using 10 μg/mL PA2024) with the final dose administered on December 31, 2012  approval, the patient consented to undergo exploratory immune monitoring as part of his subsequent retreatment with commercial sipuleucel-T. Antigen-specific antibody responses (IgG and IgM) have previously been shown to correlate with survival after treatment . Analysi9 cells/L on April 29, 2013. Unfortunately, the patient became more symptomatic from bone metastases and received palliative radiation to pubic rami and left acetabulum, completed in mid-June 2013. On June 17, 2013 PSA and WBC declined to 13.89 ng/mL and 6.2 × 109 cells/L, respectively, likely reflecting radiation effect. Most recently, the patient has been treated with enzalutamide.Four months after commercial sipuleucel-T treatment, PSA had increased to 17.38 ng/mL and WBC was stable at 8.93 × 10We report the case of an older patient with mCRPC and Rai stage 0 CLL who was treated with APC8015-2  and observed to have a dramatic and sustained PSA decline as well as improvement in his WBC. On subsequent treatment with commercial sipuleucel-T, he was also noted to have a PSA decline.This patient’s initial PSA response to treatment was noteworthy since a PSA decline of 50 % from baseline was observed in only approximately 3 % of the participants in the phase III IMPACT trial , though A second feature in this patient’s case was his concurrent diagnosis of CLL and noted decline of WBC and ALC with study treatment, suggesting the possibility of cross-reacting antigens and dual therapeutic effect of sipuleucel-T against both mCRPC and CLL. During rechallenge with commercial sipuleucel-T, his WBC was in the normal range at the start of treatment, making it difficult to interpret the effect of rechallenge on CLL. Possible explanations include that either the WBC response following initial treatment was unrelated to sipuleucel-T or that the CLL response to the initial treatment of sipuleucel-T was more durable than the PSA response.A recent report suggests that sipuleucel-T may induce humoral antigen spread . It is pOf note, active cellular immunotherapy is an approach being investigated in CLL , includiIn summary, this interesting case illustrates that the active cellular immunotherapy sipuleucel-T can result in dramatic and sustained PSA responses. In addition, treatment with sipuleucel-T can potentially lead to dual responses to other malignancies including CLL. We believe both of these remarkable findings warrant further investigation.Ethics approval was provided by the University of Washington IRB. Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Serious non-AIDS events (SNAE) are frequent in HIV patients receiving cART. Current CD4 count was shown to be more strongly associated with infective compared to not-infective SNAE and unable to predict cardiovascular events. We investigated the relationship between baseline and current CD4 count and the risk of both infective and non-infective SNAE in HIV-positive patients according to current ART use.3). Participants’ follow-up accrued from the date of enrolment (baseline) to a diagnosis of SNAE or their last visit. An event was defined the first time one of the considered SNAE occurred so that each person contributed a single event. A Poisson regression model for each of the two endpoints was used.We included all HIV-positive persons enrolled in the ICONA Foundation Study cohort who had at least one follow-up visit. SNAE were grouped in infective  and non-infective  aetiology. Incidence of these event groups were calculated overall and according to baseline and current CD4 count  and the most frequent infective SNAE were pneumonia (n=289). Crude rates of non-infective SNAE were 0.78, 1.08 and 0.80/100 PYFU, and those of infective SNAE were 1.00, 0.51 and 0.66/100 PYFU in ART naive, currently off and currently on ART patients, respectively. Higher current CD4 count was associated with reduced risk of both infective and non-infective SNAE in naives and in patients on ART . Conversely, we found no association between baseline CD4 count and risk of non-infective SNAE in people treated with ART . When CVD were considered separately, there was no association with CD4 count (not shown).A total of 10,822 patients were included  and 26.6% had hepatitis co-infections. Median age was 36 (IQR 31–42) years. Overall, 423 not-infective and 385 infective SNAE were included. The most frequent non-infective SNAE were malignancies (s on ART . The assOur findings show that, differently from ART naive, in ART-treated patients, non-infective SNAE are predicted by current but not by baseline CD4, suggesting that immune restoration is crucial to prevent these events.


This cross-sectional study assessed the psychometric properties of the Hebrew translation (PAM-H) of the PAM-13.A nationally representative sample of 203 Hebrew-speaking Israeli adults answered the PAM-H, PHQ-9 depression scale, SF-12, and Self-efficacy Scale via telephone.t 3.75; P = 0.001).Mean PAM-H scores were 70.7±15.4. Rasch analysis indicated that the PAM-H is a good measure of activation. There were no differences in PAM-H scores based on gender, age or education. Subjects with chronic disease scored lower than those without. Scores correlated with the Self-efficacy Scale (0.47), Total SF-12 (0.39) and PHQ-9 , indicating concurrent validity. Discriminant validity was reflected by a significant difference in the mean PAM-H score of those who scored below 10 (72.1±14.8) on the PHQ-9 (not depressed) compared to those scoring ≥10 (i.e. probable depression) (59.2±15.8; The PAM-H psychometric properties indicate its usefulness with the Hebrew-speaking Israeli population.PAM-H can be useful for assessing programs aimed at effecting changes in patient compliance, health behaviors, etc. Researchers in Israel should use a single translation of the PAM-13 so that findings can be compared, increasing understanding of patient activation. Hibbard and her colleagues conceptualized patient activation as encompassing a range of elements important for self-management that extend beyond any single health behavior The PAM-13 and its underlying conceptualization can help healthcare providers understand patients' strengths or weaknesses in optimizing their healthcare and health status. It has been associated with improved patient compliance and health outcomes The PAM-13 has been translated and validated in several languages including Dutch The current study assessed the psychometric properties of the Hebrew translation (PAM-H) of the PAM-13. It was hypothesized that the PAM-H would: (a) have good internal consistency and that Rasch analysis would confirm its value as a measure of activation; (b) correlate positively with measures of well-being and self-efficacy; and (c) correlate negatively with depressive symptoms.This cross-sectional study included a nationally representative sample of 203 Hebrew-speaking Israeli adults 25 to 80 years of age, with no exclusion criteria.The PAM-13 is a self-report questionnaire with 13 items. Raw scores range from 13-52. These are converted to “activation scores” ranging from 0–100 To consider concurrent validity of the PAM-H the following instruments were used:The PHQ-9 is a self-report questionnaire for screening for symptoms of depression based on the Diagnostic and Statistical Manual of Mental Disorders-IV. It has a sensitivity of 93% and specificity of 85% in primary care settings The SF-12 is a 12-item questionnaire that measures an individual's subjective sense of well-being. Scores range from 0 to 100, with higher scores indicating better quality of life Self-efficacy is an individual's belief in his/her ability to conduct specific activities. Measures of self-efficacy have been shown to predict behavior In the current study, the PAM-13 was translated as recommended by the World Health Organization for cross-cultural adaptation of self-report measures The sample design and data collection were conducted by the Cohen Institute for Public Opinion Research at Tel Aviv University. A probabilistic stratified sampling of households was built based on the official statistical areas classified according to socio-demographic characteristics. Areas were then matched with the computerized list of subscribers to the national telephone company and households randomly chosen. The proportion of Israeli households that possess landline telephones is 85%.Potential participants were contacted by telephone. If the person who answered met study criteria , the purpose of the research, expected length of interview and nature of participation (responding to questionnaire items and providing basic demographic data) were described. The person was then specifically asked if he/she was willing to participate and if verbal informed consent was granted, then the interviewer proceeded with the telephone interview. Only one adult in each household was interviewed. No personal identification  was requested or recorded. Participant consent was recorded by the interviewers and evidenced by proceeding with the interview upon receipt of consent. When consent was not received (in 153 cases), these were noted as ‘refusals’ and so reported. The interviews were conducted during December 2013.The sample size was determined using the Epi Info program, v.6 . Based on the results of an earlier Israeli study Data were entered onto an Excel worksheet and analyses were conducted using SAS Version 9.2 for SOLARIS software. The PHQ-9 scores were analyzed both as continuous and dichotomous variables. The PHQ-9 scores were dichotomized into 0-9 (not depressed) and ≥10 (probable depression), where indicated. Discrete variables were analyzed by chi-square test and continuous variables by Pearson Product Correlation or t-test for difference in means. Mean scores were compared by t-test, or by GLM procedure for variables with more than two categories. Psychometric analysis of the PAM-H was conducted using the Rasch measurement model with Winsteps software The study was approved by the Ethics Committee of Bar Ilan University. Receipt of written informed consent was not feasible in the context of this telephone-interview study based on a randomly-chosen sample of households. The researchers' description of requesting and receiving verbal informed consent (noted above) was described to the Ethics Committee on the application for approval, and approval was granted for verbal informed consent.Among 395 individuals who were contacted, 203 (51.4%) participated in the study; 153 (38.7%) declined to be interviewed and 39 (9.9%) telephones were not answered after several attempts. It was not possible to distinguish the respondents from non-respondents because this was a random population telephone survey and non-participants could not be asked for personal information.The participants were divided almost evenly between males and females, and between those with up to 12 years of education and those with more than 12 years . Almost PAM-H scores ranged from 27.9 to 100, with a mean of 70.7±15.4 . The Cronbach's alpha for internal consistency was 0.77. Rasch analysis indicated that the PAM-H is a good measure of activation There were no differences in the mean PAM-H score based on gender, age group or education . Those wSF-12 scores ranged from 17–44 (35.6±6.0) and Self-efficacy Scale scores ranged from 13–40 (34.9±4.6). PHQ-9 scores ranged from 0–20 (3.8±4.2), with 10.4% of the participants scoring 10 or above, which indicates a probable depressive episode.Concurrent validity was demonstrated by correlations in the expected direction with the three comparison questionnaires. As shown in t 3.75; P = 0.001).Discriminant validity was indicated by the significant difference in the mean PAM-H score of those who scored below 10 on the PHQ-9 (not depressed) compared to those scoring ≥10 (probable depression);  was somewhat higher than that reported in the original study of Hibbard et al. (61.9) on the development of the PAM-13 Within the study group, the mean PAM-H score was significantly lower among participants with chronic diseases compared to those without such conditions. In the present study, among those with chronic conditions, the mean PAM-H score was similar to that reported in other patient populations As expected, PAM-H scores were positively correlated with a subjective sense of well-being and self-efficacy, and negatively correlated with depressive symptoms. Although these correlations were not extremely high, they were all significant and similar to those found in other studies comparing the PAM-13 with scales measuring these constructs. For example, Hibbard et al. An earlier study of 278 patients visiting a primary health care clinic in southern Israel employed a Hebrew translation of the PAM-13 Conducting this study by means of a telephone interview could be considered a limitation of the present research. However, it should be noted that in our earlier study The Hebrew translation of the PAM-13 has been found reliable and valid for use with the Hebrew-speaking Israeli population.An important aspect of the PAM-13 is its usefulness in assessing intervention programs aimed at effecting changes in measures such as patient compliance, health behaviors


Acinetobacter baumannii; however, the mechanism underlying resistance to this synergistic combination is not well understood. In the present study, two A. baumannii isolates, AB1845 and AB2092, were isolated from a patient with hospital-acquired pneumonia before and after 20 days of cefoperazone/sulbactam therapy . The minimum inhibitory concentration (MIC) of cefoperazone/sulbactam for AB1845 and AB2092 was 16/8 and 128/64 mg/L, respectively. Blood samples were collected on day 4 of the treatment to determine the concentration of cefoperazone and sulbactam. The pharmacokinetic/pharmacodynamic (PK/PD) indices (%T>MIC) were calculated to evaluate the dosage regimen and resistance development. The results showed that %T>MIC of cefoperazone and sulbactam was 100% and 34.5% for AB1845, and 0% and 0% for AB2092, respectively. Although there was no available PK/PD target for sulbactam, it was proposed that sulbactam should be administered at higher doses or for prolonged infusion times to achieve better efficacy. To investigate the mechanism of A. baumannii resistance to the cefoperazone/sulbactam combination in vivo, whole-genome sequencing of these two isolates was further performed. The sequencing results showed that 97.6% of the genome sequences were identical and 33 non-synonymous mutations were detected between AB1845 and AB2092. The only difference of these two isolates was showed in sequencing coverage comparison. There was a 6-kb amplified DNA fragment which was three times higher in AB2092, compared with AB1845. The amplified DNA fragment containing the blaOXA-23 gene on transposon Tn2009. Further quantitative real-time PCR results demonstrated that gene expression at the mRNA level of blaOXA-23 was >5 times higher in AB2092 than in AB1845. These results suggested that the blaOXA-23 gene had higher expression level in AB2092 via gene amplification and following transcription. Because gene amplification plays a critical role in antibiotic resistance in many bacteria, it is very likely that the blaOXA-23 amplification results in the development of cefoperazone/sulbactam resistance in vivo.Cefoperazone/sulbactam has been shown to be efficacious for the treatment of infections caused by  Acinetobacter baumannii is a non-fermentative, gram-negative opportunistic pathogen that can cause hospital-acquired pneumonia (HAP), blood infections and urinary tract infection, among others  presented successful clinical efficacy  of sulbactam in A. baumannii  was applied to evaluate the dosage regimen and development of resistance. Moreover, whole-genome sequencing was employed to investigate the mechanism of cefoperazone/sulbactam resistance of the two isolates developed in vivo.In the present study, two A. baumannii (AB1845) was isolated from the sputum of the patient, revealing bacterial infection. Hence, the patient was treated with meropenem 0.5 g q8h without clinical efficacy. On day 6, the dosage regimen was changed to cefoperazone/sulbactam  for 20 days. The treatment was stopped when body temperature and white blood cell counts returned to the normal range; additionally, A. baumannii was not detected in the sputum. However, the patient had a fever, and A. baumannii (AB2092) was again isolated 4 days after cefoperazone/sulbactam treatment. The cefoperazone/sulbactam treatment was finally considered to have failed both clinically and microbiologically efficacy.This study was approved by the institutional review board of Huashan Hospital affiliated to Fudan University. Written informed consent was obtained from the patient. A 19-year-old male patient with serious brain trauma was admitted to Huashan Hospital . The patient had a high fever with white blood cell counts out of the normal range after being checked into bed for 48 h. Both chest X-Ray radiophotography and computed tomography showed pneumonia. >MIC, namely the percentage of the dosing interval for which the plasma concentration of cefoperazone or sulbactam above the MIC. It was calculated based on the drug pharmacokinetic profile and MICs of the combination , and at 0.5, 1, 2, 4, 6 h afterward on day 4  of the cefoperazone/sulbactam treatment. The drug concentration was determined using the LC-MS/MS method , which wbination .Acinetobacter baumannii AB1845 and AB2092 were isolated from the sputum of the patient before and after cefoperazone/sulbactam therapy. Both isolates were stored at -80°C. The MIC was determined in cation-adjusted Mueller-Hinton broth  using a broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) standards. The MICs of the β-lactam/β-lactamase inhibitor combination, including cefoperazone/sulbactam, carbapenems, colistin, and polymyxin B, were determined.1. Genome assembly was performed using Velvet (Ver 1.0.15) (A. baumannii MDR-ZJ06 genome (from the phylogenetic tree) using Bowtie2 with default settings  according to the instruction manual and stored at -80°C before sequencing. A 300-bp paired-end library was constructed for the purified DNA sample following the standard Illumina paired-end protocol. Cluster generation was performed in C-bot, and sequencing was performed on the Illumina Hiseq2500 with 150 cycles. The sequence reads were cleaned using the FASTX toolkit 1.0.15) . Putativ 1.0.15)  and Z-Cu 1.0.15)  program. 1.0.15)  by conca 1.0.15) . Gene ansettings . Single settings , and thesettings .blaOXA-23 was quantified by real-time PCR. AB1845 and AB2092 were cultured in cation-adjusted Mueller-Hinton broth and collected during the early log phase. Total RNA was extracted using a TaKaRa Mini BEST Universal RNA Extraction Kit  according to the manufacturer’s instruction. Quantitative real-time PCR (qPCR) was performed using a two-step process. RNA was first reverse-transcribed to cDNA , and real-time PCR was conducted on an ABI Vii7  using a TaKaRa SYBR® FAST qPCR Kit with 40 cycles of denaturation for 5 s at 95°C, annealing for 30 s at 50°C, and extension for 20 s at 72°C. The PCR primers for the blaOXA-23 gene were F: CCGAGTCAGATTGTTCAAGGA and R: TGTAGAGGCTGGCACATATTC; and those for 16S rRNA were F: GGCGGCTTATTAAGTCGGATG and R: TTCGTACCTCAGCGTCAGTATT. The melting curve analysis was performed immediately after amplification to verify the specificity of the PCR amplification products.The mRNA expression level of ΔΔCT method . The relative quantitation was calculated by the 2-T method . The stuTable 1). The MICs of the cefoperazone/sulbactam combination were 16/8 mg/L for AB1845; and the MICs of mono cefoperazone and sulbactam were >128 mg/L and 32 mg/L. The results supported a synergistic effect of the combination . However, the MICs of the combination and mono drugs were 128/64, >128, and 128 mg/L for AB2092. The combination did not display a synergistic effect on AB2092.Minimum inhibitory concentrations were obtained for cefoperazone/sulbactam combinations, carbapenems, colistin and polymyxin B for the pre-therapy isolate AB1845 and the post-therapy isolate AB2092 . The isolates also developed resistance to imipenem and doripenem. Since both cefoperazone and meropenem are β-lactam antibiotics, the increased MIC in both cefoperazone/sulbactam and carbapenems may imply the similar underlying resistance mechanism. The isolates were only sensitive to colistin and polymyxin B, indicating an alternative choice for the AB2092 treatment. The MICs of the antibiotics are shown in Figure 1). The pharmacokinetic parameters of cefoperazone and sulbactam are summarized in Table 2. The T1/2 of cefoperazone was 4.1 h, which was a bit longer than the reported 1.8 h , and raw sequence reads for AB1845 and AB2092 have been submitted to NCBI’s Sequence Read Archive under the study accession number SRP072783.The two isolates were initially typed by PFGE of Figure 2). The 3,718 CDSs were homologous  to the genome of A. baumannii MDR-ZJ06, which was a multi-drug-resistant isolate belonging to International clone II and wide spreading in China . The results showed that the two isolates possess 97.6% similarity to MDR-ZJ06, with 921 and 915 SNPs, respectively. Certain genes were missing in both AB1845 and AB2092 compared with MDR-ZJ06. For example, 17 genes had a 15-kb fragment that was missing in the resistant island AbaR22, which includes transposition helper proteins, site-specific tyrosine recombinase, transposase protein A and conserved hypothetical proteins, among others.Automated gene prediction detected 3,811 and 3,810 putative coding sequences (CDSs) for AB1845 and AB2092, respectively. A phylogenetic tree was constructed, and the genome similarity was compared .A comparison of the CDS of AB1845 and AB2092 revealed only 33 SNPs. All of these SNPs were synonymous, indicating that SNP was not the main reason for the development of resistance (blaOXA-23 and blaOXA-51) β-lactamase were identified in both AB1845 and AB2092 (Supplementary Table S2).A well-known resistance enzyme for β-lactam antibiotics is β-lactamase. The class A, C, and D classes (Supplementary Table S3). These six genes were AAA ATPase, DEAD/DEAH box helicases, blaOXA-23, and three hypothetical genes. Among these genes, AAA ATPase and DEAD/DEAH box helicases are involved in the function of DNA replication, repair, transcription, and two uncharacterized genes .Gene amplification was detected by whole-genome sequencing in combination with the read depth method . Approxied genes . The blaperazone . All sixn AB2902 . The ampblaOXA-23 in AB1845 and AB2092. Both isolates were collected at the early log phase, and the qPCR results showed that the mRNA level of blaOXA-23 was five times higher in AB2092 than in AB1845 . This finding was consistent with the gene amplification results.Quantitative real-time PCR was conducted to measure the mRNA levels of Acinetobacter baumannii commonly colonizes in respiratory tract of the hospitalized patients . However, the reads depth method showed six amplified genes on reported . The traisolates . Tn2008 in China . IS elemlaOXA-23 , which cc stress . In a st numbers . Recent mrD gene . Numerouin vitro . During ribosome . Anotherethoprim .blaOXA-23 in the isolates is very likely the contribution to the resistance. It is rational to conferred resistance to carbapenem antibiotics by the amplification of blaOXA-23. However, this is the first time to report the amplification of blaOXA-23 results in cefoperazone/sulbactam combination resistance. Further studies will be focused on collecting more clinical isolates pairs of A. baumannii to support the mechanism proposed.In this study, the isolates developed resistance to cefoperazone/sulbactam as well as carbapenem antibiotics. The whole-genome sequencing showed the amplified XL designed the genomic sequencing experiments. XL and HZ performed the data analysis. XL and ZS conducted the qPCR experiments. YC conducted the pharmacokinetic parameter calculation. LS extracted the genomic DNA for sequencing. MZ conducted the MIC test and the PFGE test. JS and JZ conceived of the study and devised the overall study strategy. XL wrote the manuscript with input from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clostridium difficile is an opportunistic pathogen inhabiting the human gut, often being the aetiological agent of infections after a microbiota dysbiosis following, for example, an antibiotic treatment. C. difficile infections (CDI) constitute a growing health problem with increasing rates of morbidity and mortality at groups of risk, such as elderly and hospitalized patients, but also in populations traditionally considered low-risk. This could be related to the occurrence of virulent strains which, among other factors, have high-level of resistance to fluoroquinolones, more efficient sporulation and markedly high toxin production. Several novel intervention strategies against CDI are currently under study, such as the use of probiotics to counteract the growth and/or toxigenic activity of C. difficile. In this work, we have analyzed the capability of twenty Bifidobacterium and Lactobacillus strains, from human intestinal origin, to counteract the toxic effect of C. difficile LMG21717 upon the human intestinal epithelial cell line HT29. For this purpose, we incubated the bacteria together with toxigenic supernatants obtained from C. difficile. After this co-incubation new supernatants were collected in order to quantify the remnant A and B toxins, as well as to determine their residual toxic effect upon HT29 monolayers. To this end, the real time cell analyser (RTCA) model, recently developed in our group to monitor C. difficile toxic effect, was used. Results obtained showed that strains of Bifidobacterium longum and B. breve were able to reduce the toxic effect of the pathogen upon HT29, the RTCA normalized cell-index values being inversely correlated with the amount of remnant toxin in the supernatant. The strain B. longum IPLA20022 showed the highest ability to counteract the cytotoxic effect of C. difficile acting directly against the toxin, also having the highest capability for removing the toxins from the clostridial toxigenic supernatant. Image analysis showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight-junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity. Clostridium difficile is a Gram-positive, spore-forming, motile and strict anaerobe rod that can be found in the gastrointestinal tract of humans and animals  is the main cause of diarrhea associated with antibiotic use or related to health-care environments  strain. This method is based on the continuous monitoring of the impedance signal, transmitted through gold microelectrodes placed in the bottom of microtiter plates, of HT29 monolayers  intestinal epithelial cell line HT29, to follow in real time the effect of supernatants collected from nolayers . Our aimBifidobacterium and Lactobacillus species used in this study are listed in Table 1. Most strains belonging to IPLA culture collection were isolated from infant feces and breast milk ] at 37°C in the anaerobic chamber MG500  under 80% N2, 10% CO2 and 10% H2 atmosphere. As standard procedure bacterial stocks, kept at -80°C in MRSC + 20% glycerol, were spread onto the surface of agar-MRSC and incubated for 3 days. A single colony was picked to inoculate MRSC broth which, after 24 h incubation, was used to inoculate (2%) 10 ml fresh MRSC broth. This culture was incubated overnight (18 h) to prepare the bacterial suspensions that will be described next.The ast milk , whereasast milk . StrainsC. difficile LMG21717  producing both TcdA and TcdB toxins (Toxinotype 0) was purchased from the “Belgian Coordinated Collections of Microorganisms” . The strain was routinely grown in Reinforced Clostridium Medium  in Hungate tubes under anaerobic conditions at 37°C. Frozen stocks (-80°C in RCM + 20% glycerol) were directly activated in RCM broth incubated for 24 h and this culture was used to inoculate (2%) fresh medium that was cultivated for 13 h. This culture was used as inoculum to obtain the toxigenic supernatant.The strain C. difficile LMG21717 have previously been determined and published  to obtain the C. difficile-free toxigenic supernatant (Tox-S), which was kept in several aliquots at -80°C.Conditions to obtain toxigenic supernatant from ublished . In shorTwo independent ELISA tests  were used to quantify the concentration of TcdA or TcdB in the toxigenic supernatant, as well as the remnant toxins in the neutralized bacterial-supernatants obtained after incubation of Tox-S with bifidobacteria and lactobacilli.Supplementary Figure S1A. Bifidobacteria and lactobacilli cultures grown for 18 h in MRSC were washed twice with PBS and resuspended at 109 cfu/ml in the HT29-cultivation medium  supplemented with 5% of Tox-S from C. difficile or without supplementation (controls). After incubation for 1 h under anaerobic conditions and mild stirring (∼300 rpm), the bacterial suspensions were centrifuged  to obtain bifidobacteria- or lactobacilli-free bacterial supernatants. Then, the pH was increased to 7.55 ± 0.05 with 1 and 0.1 N NaOH and the volume obtained was adjusted to twice the initial one with MM; this means that the maximum amount of remnant toxin that could be present was 2.5%. These neutralized cell-free supernatants (NCFS) were directly used to test their cytotoxicity upon HT29 monolayers as well as to quantify the remnant TcdA and TcdB toxins. This screening was performed with two biological replicates, each analyzed in duplicate, of each bacterial strain using HT29 monolayers of two consecutive passages (p147 and p148).The experimental design carried out in this study is schematized in B. longum IPLA20022 was selected in order to determine whether the capability to diminish the cytotoxic effect of C. difficile supernatant was dependent on bacterial viability. For that purpose UV-treated IPLA20022 suspensions were prepared from MRSC-grown cultures that were washed and resuspended in PBS at 109 cfu/ml. Then, the PBS suspension was poured into several petri dishes allowing a high surface spread and they were submitted to ultra violet radiation in a UV-chamber . Three UV cycles of 30 min were applied, homogenizing the PBS suspension in each interval, and the absence of viability was checked by plating serial dilutions of UV-treated IPLA20022 suspension in agar-MRSC (C. difficile supernatant was performed as previously described. A non UV-killed suspension (live IPLA20022) of the same culture was used as control. After incubation for 1 h, both suspensions were processed to obtain the respective NCFS . This experiment was carried out with three independent cultures  of strain IPLA20022 upon HT29 monolayers within the same passage (p149), each measured in duplicate.The strain gar-MRSC . IncubatB. longum IPLA20022 against C. difficile toxins, cell-free bifidobacterial supernatants obtained from three independent-culture replicates  were incubated with 50% toxigenic (Tox-S) supernatant for 1 h under anaerobic conditions. Afterward, supernatants were neutralized (pH ≥ 7.5) and its cytotoxic activity tested upon HT29 monolayers (passage p149) at 2.5% in MM .To test the activity of putative secreted factors by 2. McCoy’s Medium (MM) supplemented with 10% foetal bovine serum (FBS), 3 mM L-glutamine and a mixture of antibiotics  was used for HT29 cultivation. The pH value of supplemented MM was 7.48 ± 0.02. All media and reagents were purchased from Sigma–Aldrich. Maintenance of the cell line, between passages 145 to 149, was performed under standard conditions, at 37°C 5% CO2 atmosphere, in a CO2-Series Shel-Lab incubator .The intestinal cell line HT29 (ECACC 91072201), from human colon adenocarcinoma, was purchased from the “European Collection of Cell Cultures”  and stored at IPLA under liquid N2 atmosphere. This technology records variations in impedance due to the adhesion, growth and morphological changes of HT29 cells during interaction with gold-microelectrodes placed in the bottom of specific microtiter plates (E-plates). The impedance signal is converted in the arbitrary “cell index” (CI) unit which is recorded in the external computer allowing, as well, data analyses through the RTCA software 1.2.1 (ACEA Bioscience).The real time cell analyzer (RTCA-DP) xCelligence  used to monitor HT29 cells performance upon the different conditions tested, was introduced in a Heracell-240 Incubator  set at 37°C with 5% COC. difficile toxins was previously described by 5 HT29 cells (in 100 μl) and monitored  for 22 h to ensure the formation of a monolayer (confluent state). Afterward, the medium was removed and 200 μl of the different bacterial NCFS were added per well. Additionally, wells containing 200 μl of a control without bacteria but with Tox-S  or 200 μl MM medium without bacteria or Tox-S added (non-cytotoxic control) were included in each experiment. The monitoring continued (every 10 min) for an additional 20–22 h under standard incubation conditions. CI values recorded were normalized by the time of the supernatant addition and by the control sample (MM) as previously described (The method to monitor the damage caused by escribed . Samples6 HT29 cells/ml (1 ml) and placed on top of the microscope objective. Images were recorded every 15 min until the confluent state was reached (about 22 h); afterward, culture medium was removed and 1 ml of fresh medium containing 2.5% Tox-S or 1 ml of the NCFS collected after incubation of live B. longum IPLA20022 with Tox-S, was added in two independent μ-Slides. Image capture was performed for additional an 16 h.Several images were captured in real time using the compact, inverted, optical microscope (40× objective) LumaScope-600 Series  which was placed inside the Heracell-240 incubator. Images were recordered in an external computer with the software LumaView600Cy 13.7.17.0 . To this end, 2-well μ-Slide  were seeded with 2 × 106 HT29 cells/ml (0.3 ml) and incubated for 22 h to reach confluent state. Afterward, supernatant was removed and wells (in duplicate) were filled with the same volume of fresh medium containing MM (control), 2.5% Tox-S, and NCFS from live or dead B. longum IPLA20022 incubated with Tox-S. Incubation continued for additional 20 h; then, supernatant of each well was removed and HT29 monolayers fixed with 1 vol (0.3 ml) of cold (-20°C) acetone for 10 min. Samples were washed twice with PBS for 5 min under mild stirring and permeabilised with PBS containing 0.1% Triton 100x (Sigma) for 15 min. The nonspecific binding sites were blocked with FBS (25% in PBS) for 20 min and finally washed once with PBS. The Phalloidin-Alexa-Fluor-568 probe  toward F-actin was added in 0.3 ml of PBS  and samples were incubated overnight at 4°C in darkness. After washing twice with PBS, HT29 nucleus were stained with DAPI probe  used at 1:1000  and incubated under the same conditions for, at least, 6 h. Finally, samples were washed and added to 0.3 ml of PBS previous visualization under microscope.HT29 monolayers submitted to different treatments were analyzed by confocal scanning laser microscopy (CSLM) after an end-point incubation period of 20 h. For this, 8-well μ-Slide  were seeded with 2 × 10Z-stacks of HT29 samples were acquired using a 63x/1.4 oil objective applying a line average of 2 to reduce noise on the final images and a z-step of 1 micron. Details of a region were later acquired using a 2.50 optical zoom. Image-captures were recorded with the “Leica Application Suite X” software version 1.8.1.13759 (Leica).For the CSLM analysis the Leica TCS AOBS SP8 X confocal microscopy  located in the Scientific-Technical Services of Oviedo University, was used. DAPI and Alexa-Fluor-568 fluorochromes were excited at 405 nm by a blue–violet laser diode and at 578 nm by a white light laser, respectively. B. longum IPLA20022, one-way ANOVA followed by SNK  mean comparison tests were performed. The statistical package IBM SPSS Statistics for Window Version 22.0  was used to carry out these analyses. Legend of Figure 4 describes the comparisons made in each type of experiment.To assess differences in the response  of HT29 due to the anti-toxin activity of C. difficile upon intestinal cell lines was used to address the anti-toxin probiotic potential of twenty bifidobacteria and lactobacilli strains. As an initial step several parameters were optimized in order to establish conditions for the screening using as a biological model confluent-HT29 monolayers (data no shown). Finally, neutralized (pH ≥ 7.5) cell-free supernatants (NCFS), obtained after incubation  of each strain (about 1 × 109 cfu/ml) with 5% C. difficile supernatant (Tox-S), were used for this study . The behavior of HT29 monolayers was monitored in real time recording the variations in the impedance signal  over time due to the presence of the NCFS, the toxigenic control (2.5% Tox-S), or the culture media alone . To understand the impedance graphs is worth noting that the lowest normalized-CI value indicates the highest toxigenic capability of C. difficile supernatant upon HT29; thus, in Figure 1, the red line  is the control for damage, whereas the pink line represents the non-toxigenic control (MM) used as a reference for normalization of all CI values being the reason to have “0 value.” Regarding the effect of NCFS, those obtained after incubation of strains in MM medium without C. difficile toxins (dotted lines) showed normalized-CI values equal or higher to the control, therefore indicating the absence of any toxic effect induced by the putative probiotics. However, when the NCFS obtained from bacteria incubated with Tox-S were analyzed, HT29 monolayers behaved differently depending on the species considered . Graphics obtained clearly show that strains belonging to species Bifidobacterium bifidum and B. animalis subsp. lactis had no protective effect against C. difficile toxins since the normalized-CI lines obtained showed similar, or even lower, values than the toxigenic Tox-S control. By contrast, the normalized-CI obtained from the four lactobacilli tested, as well as the strains of B. longum, B. breve, and B. pseudocatenulatum were higher than those induced by C. difficile supernatant. In general, normalized-CI lines from B. longum and B. breve strains were the closest to the control, thus being the strains showing higher anti-toxin capability.The method previously developed by our group to detect in real time the toxic effect of Table 1). Results obtained in the short term showed that all strains belonging to B. longum and B. breve, as well as L. gasseri IPLA20121, L. paracasei IPLA20124 and L. rhamnosus GG, seemed to have higher values of normalized-CI than the toxigenic control. However, none of the lactobacilli were able to keep the protective effect upon HT29 for a prolonged period (22 h). The strain C. difficile LMG21717 used in this study produced about ten-times more TcdA than TcdB (Table 1) and the strains showing high protective effect were those that apparently were more effectively in reducing the concentration of TcdA, i.e., belonging to B. longum and B. breve species (Table 1). Indeed, the NCFS obtained from strain B. longum IPLA20022 that promoted the lowest damage after 22 h only had 12% of remnant TcdA. Of note is that NCFS from B. bifidum and B. breve seemed to have a good ability to reduce TcdB levels (remnant between 23 and 44%), although this fact was not correlated with higher protective effect in B. bifidum because this species seemed to be less effective against TcdA.Normalized-CI obtained 4 h after NCFS addition (short term effect) or 22 h after (long term effect) were analyzed in more detail  showed that HT29 cells treated with 2.5% toxigenic C. difficile supernatant become spherical and the integrity of the monolayer was lost when incubation was prolonged . However, monolayers added with NCFS from live B. longum IPLA20022 incubated with Tox-S remained more stable and only after a long incubation period (16 h) some cellular particles were released to the culture medium . Furthermore, although the cytopathic mechanism of C. difficile toxins is well known, we performed immunohistochemistry CSLM analysis to confirm the cellular events under different treatments . Control HT29 monolayers (grown in MM for 20 h) showed a typical F-actin cytoskeleton in which the nucleus is imbibed, thus having an epithelial-like morphology with annexed cells well connected. However, monolayers treated for the same period with Tox-S supernatant lost the interconnection among F-actin filaments  and the nucleus seems to be in the initial stages of apoptosis, i.e., the chromatin initiates the condensation showing more intense blue due to DAPI staining ; therefore, HT29 cells become more spherical  and it seems that the tight junctions that maintain the monolayer integrity might have been disrupted . The photographs obtained from HT29 monolayer treated for 20 h with the NCFS from live B. longum IPLA20022 were more similar to the control without toxin; the F-actin cytoskeleton still showed an interconnected structure and the nucleus showed less intense DAPI staining comparable to that of the negative control than the toxigenic control. This structure, resembling that of intact epithelial monolayers, is in agreement with the presence of lower amounts of remnant toxin in the NCFS and higher normalized-CI due to the capability of this strain to counteract the effect of clostridial toxins.Time-lapsed microphotographs . Indeed, the statistical analysis performed at 4 and 22 h after NCFS addition showed that live IPLA20022 had a significantly (p < 0.05) higher normalized-CI, i.e., higher protective capability, than the dead strain and the toxigenic control . Consequently, the immunohistochemistry study confirmed that the morphology of HT29 treated for 20 h with the NCFS from dead B. longum IPLA20022 was more similar to that obtained with the toxigenic control . Indeed, besides the F-actin modification, some apoptotic bodies were evidenced in both toxigenic and dead-IPLA20022 samples  suggesting that the UV treatment of this strain, which probably affected the structure and function of the cell envelope, abolished the anti-clostridial effect of B. longum IPLA20022. Finally, we have tested the activity against clostridial toxins of the supernatants obtained from overnight cultures of this bifidobacterial strain. Surprisingly, the normalized-CI values were similar to those of those obtained with (live) pellets and both of them were statistically (p < 0.05) higher than the toxigenic control . This result suggests that B. longum IPLA20022 is able to secrete factors having activity against the toxins of C. difficile.In order to determine whether this strain retains its anti-toxin capability under non-viable conditions, C. difficile strains . The method used, based on impedance measurement of HT29 monolayers  with twenty five bifidobacteria or lactobacilli and they found that the capability to antagonize the toxic effect upon Vero line (monkey fibroblast-like kidney cells) was strain dependent, but they did not report a species-efficacy association. Nevertheless, the experimental procedure used in our screening for detecting anti-toxicity was based on the incubation of the probiotic strains with a toxigenic supernatant from C. difficile, previous to analyze the effect of NCFS upon the biological model HT29. Then, a priori, the putative mechanisms that could be behind the anti-toxin capability detected with our approach are the modification of the C. difficile toxin and/or its availability for acting on the epithelial cells.In our study, we have explored the capability of twenty lactobacilli and bifidobacteria to counteract the effect of toxins (TcdA and TcdB) from nolayers , allowedC. difficile toxins. Lactobacillus delbrueckii subsp. bulgaricus B-30892 releases bioactive components, of unknown nature, able to decrease the toxic effect of C. difficile ATCC9689 upon epithelial intestinal Caco2 cells. Similarly, Lactococcus lactis subsp. lactis CIDCA8221 secretes heat-sensitive products, higher than 10 kDa, that are not affected by treatment with proteases or protease-inhibitors, which were able to protect Vero cells from C. difficile toxins  found in the genome of B. longum  decreased the tcdA expression and toxin-A release. In vivo (CDI mouse model) treatment with live S. thermophilus showed a significant inverse correlation between levels of luminal lactic acid and C. difficile abundance in the murine gut, thus reducing the disease activity indexes of experimentation animals  produced in this short incubation period by lactobacilli or bifidobacteria was neutralized.Regarding the adsorption as mechanism to reduce toxins activity, it has been demonstrated that the soluble S-layer protein from the surface of e toxins . Howeverne model . The lac animals . In our B. longum and B. breve strains (pointing to a species-dependent efficacy) suggests that some specific characteristics of these two phylogenetically close species (C. difficile toxicity. Another interesting observation that will deserve further attention is the (apparently) better capability of B. bifidum, and to a lower extent of B. breve, to specifically reduce TcdB levels.Based on the results describe in this article, the adsorption of toxins to the bifidobacterial surface as well as the presence of secreted molecules responsible for the anti-toxigenic effect observed, are both plausible mechanisms of action. Nevertheless bacterial viability, which may be also needed to keep a functional bifidobacterial envelope, is required in order to maintain the anti-clostridial activity. Finally, the highest anti-toxin capability of  species  could acC. difficile. The impedance-based, RTCA xCelligence was a fast, reliable and efficient method for the screening of a large collection of bacteria allowing the selection of those strains with higher protection capability. In our case, strains from B. breve and B. longum showed the better performance, since they were able to reduce the levels of toxins from C. difficile supernatants. The best candidate to be used as probiotic to alleviate CDI was B. longum IPLA20022; this was the strain with the highest in vitro capability for reducing the levels of clostrial toxins, as well as for avoiding the cytopatic effect upon the intestinal epithelial cellular line HT29. Apart for elucidating the mechanism behind this anti-toxigenic capability, the next steps will be to study the efficacy of B. longum IPLA20022 in more complex in vitro and in vivo biological models before proposing its human application to treat CDI.In this work we have optimized a protocol to search for potential probiotics with anti-toxic activity against toxins synthesized by MG and PR-M contributed with the conception, experimental design and results interpretation of this study. LV-V carried out all experiments, OG-S advised the immunohistochemistry analysis and MA-G perform the CSLM analysis. PR-M was in charge of writing the drafted manuscript. All authors performed a critical revision of the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


About half of all carious lesions in primary teeth of German 6- to 7-year-old children remain untreated, but no data regarding the clinical consequences of untreated dental caries are available. Therefore, this cross-sectional observational study aimed to assess the prevalence and experience of caries and odontogenic infections in the primary dentition of 5- and 8-year-old German children.n = 496) and in 8-year-old primary school children (n = 608) living in the Westphalian Ennepe-Ruhr district. Schools and preschools were selected by sociodemographic criteria including size, area, ownership, socio-economic status. Caries was recorded according to WHO criteria (1997). The Lorenz curves were used to display the polarisation of dental caries. Caries pattern in 5-year-olds was categorized by Wyne’s (1997) definition of early childhood caries (ECC). Odontogenic infections as clinical consequence of untreated dental caries were assessed by the pufa index. The ‘untreated caries-pufa ratio’ was calculated, and the Spearman’s rank correlation coefficient (ρ) was used for evaluating the correlation between dmft and pufa scores. Categorical data were compared between groups using the chi-square test and continuous data were analysed by t-test.Dental examinations were performed in 5-year-old pre-school children  was the prevalent caries pattern in 5-year-olds. About 30 % of the tooth decay was treated (5y: 29.7 %/8y: 39.3 %). The Lorenz curves showed a strong caries polarisation on 20 % of the children. Pufa prevalence and experience was 4.4 %/0.1 ± 0.5 pufa in 5-year-olds and 16.6 %/0.3 ± 0.9 pufa in 8-year-olds. In 5-year-olds 14.2 % and in 8-year-olds 34.2 % of the d-component had progressed mainly to the pulp. A significant correlation between dmft and pufa scores exists in both age groups . First deciduous molars were most frequently affected by odontogenic infections, presenting virtually all pufa scores (>95 %).Prevalence and experience of odontogenic infections and the untreated caries-pufa ratio were increasing from the younger to the elder children. Dmft and pufa scores in primary teeth predict a higher caries risk in permanent teeth. The pufa index highlights relevant information for decision makers to develop effective oral health care programs for children at high risk for caries. Dental caries continues to be one of the most widespread diseases in the world . In partSeverely decayed teeth have an important impact on children’s general health, nutrition, growth and body weight –10 by caThe early onset of dental caries is of exceptional concern since it represents an indicator for missed opportunities for preventive care and endangers the general health of a child. Wyne classified dental caries in children aged younger than 6 years as early childhood caries (ECC) by three types of severity . Type I teeth indicated for extraction’ is a WHO criterion for treatment needs [Most epidemiological studies performed in children have used the dmft index, which provides information on the caries experience and restorative and surgical treatment, but fails to contribute data on the consequences of untreated caries. The diagnosis ‘nt needs , but doeIn 2010, Monse et al.  introducRecently, the pufa index was used particularly in low- and middle-income countries to display the severity of oral health neglect Table . The pufn = 496) and eight (n = 608) years living in the EN District in Germany were included in this cross-sectional epidemiological study. By the law, free dental screenings to all children and adolescents attending preschools and schools are annually offered by the Department of Social Services and Health, Health Services for Children and Adolescents of the EN District. The age group of 5-year-olds was chosen because of a good comparability to international studies regarding the pufa index  in Western Germany. NRW experienced high industrialisation and urbanisation in the early 20th century, becoming the largest conurbation and centre of coal industry in Europe. Because of the coal-crisis in the years following 1960, the socio-economic status declined. In 2011 NRW had an at-risk-of-poverty rate of 14.6 % which is comparable to Germany (15 %) . The repThe sample size was estimated to the number of children necessary to obtain statistical significance with 80 % power and an interval of 5. Targeting the estimated sample size and following the regional socio-demographic pattern, 21.0 % (34 out of 162) pre-schools and 38.2 % (21 out of 55), primary schools were selected from the different areas. Selection criteria of the schools/pre-schools included area , size , ownership (public/private) and socio-economic status targeting a proportional distribution.The exclusion criteria for the cross-sectional study were: 1) absenteeism from school/pre-school 2) child has a special health care need and 3) refusal of the child to be examined in the pre-school setting. Eighty per cent (495 out of 620) of all 5-year-olds attending the selected pre-schools and 88.9 % (608 out of 684) of all 8-year-olds attending the selected primary schools could be included in this study.Prior to the survey, the examiner (I. G.) received 1-day theoretical and clinical calibration training for using the dmft and pufa indices. An experienced dentist and epidemiologist (R. H.-W.) conducted the training. Ten children were examined in a pre-school and a primary school not included in this survey, but under the same field conditions as in the main study. The intra- and inter-examiner-reproducibility was assessed by the kappa (κ) statistics. The κ values for inter-examiner-reproducibility ranged from 0.90 (I. G.) to 0.92 (R. H-W.) for the pufa index, demonstrating excellent agreement, and values for inter-examiner reproducibility for the dmft was in the same range (0.88 I. G/0.93 R. H-W.). Intra-examiner reproducibility ranged from 0.89 to 1.00 for the examiner (I. G.) for both indices. Within the main study, every 20th child was repeatedly examined. The intra-examiner reproducibility for both indices ranged between 0.91 and 1.00.The examinations were performed by one calibrated dentist in classrooms in each pre-school or primary school (I. G.). The caries status of the children was assessed according to WHO criteria  using anThe clinical consequences of untreated caries were recorded by using the pufa index . The pufp ≤ 0.05.Data collection was performed with excel spreadsheets  and the statistical analysis of the oral health data was carried out using SPSS 21.0. , R 3.1.1 . CariesThis study was performed in full accordance with ethical principles and approved by the ethics committee of the Jena University Hospital (registration number 3660-D1/13).p = 0.035) and also had a higher untreated caries-pufa-ratio . First primary molars were affected most frequently by odontogenic infections .Four hundred and six 5-year-olds 249 boys) with an average of 19.4 primary and 0.7 permanent teeth were included in the analysis. Caries prevalence was 26.2 % and caries experience was 0.9 ± 2.0 dmft (Table 49 boys wSix hundred eight 8-year-olds 298 boys) with 11.3 permanent and 11.7 primary teeth were examined. Caries prevalence was 48.8 % in the primary . The prevalence of caries experience and odontogenic infections was significantly higher in boys. Almost 40 % of untreated caries in boys revealed a pufa index >0. Figure p = 0.00) and 34.9 %   , 29. ThiUntreated caries may affect seriously the quality of children’s life because of pain and discomfort, which could lead to acute and chronic infections, oral mucosal conditions and altered eating and sleeping habits , 39. Furρ = 0.399, p < 0.001). Hence, the second null hypothesis that untreated dental caries does not correlate with odontogenic infections was rejected.About one-third to one-half of Westphalian 5- to 8-year-olds suffered from caries in primary teeth, which is in the same range 47.3 %) estimated for 6- to 7-year-olds in the last representative epidemiological German study in 2009 .3 % esti. CommonlTaking into account the different socio-economic background of the population examined, comparisons with other countries may be limited. The pufa prevalence of our population was considerably low  compared to Filipino 6-year-olds (85 %) , BraziliConsistent with the studies mentioned, pulp involvement (p) was the diagnosis most frequently recorded, followed by ulceration (u) especially in 8-year-olds. This is in contrast to the findings of Figueiredo et al.  and BagiFrigueiro et al.  and MurtThe use of only the dmft/DMFT index may be misleading the interpretation of caries epidemiological data. That was shown by the national oral health survey of the Philippines  reportinDental caries is a multifactorial chronic disease with the interplay of individual, cultural, social and socio-economic risk factors. The lack of data regarding these factors is a limitation of the present cross-sectional study. Nevertheless, using the pufa index provides a more comprehensive view on caries pattern in primary teeth of German children. However, there is a lack of other studies performed in high-income countries to compare the findings.This is the first German survey showing prevalence and experience of odontogenic infections as consequences of severe untreated dental caries in primary teeth among German 5- and 8-year-olds by using the pufa index. Prevalence and experience of odontogenic infections and the untreated caries-pufa ratio were increasing from the younger to the elder children. Pufa scores in primary teeth predict a higher caries risk in permanent teeth. The pufa index highlights relevant information by assessing the severity of untreated dental caries for dentists and decision makers to develop effective oral health care programs for children at high caries risk.